4.1B suppresses cancer cell proliferation by binding to EGFR P13 region of intracellular juxtamembrane segment

Background Gastric cancer (GC) has high incidence and mortality worldwide. However, the underlying mechanisms that regulate gastric carcinogenesis are largely undefined. 4.1B is an adaptor protein found at the interface of membrane and the cytoskeleton. Previous studies demonstrated that 4.1B serves as tumor suppressor. Results We showed that 4.1B expression was decreased or lost in most GC patients. The expression pattern of it was tightly correlated with tumor size, TNM stage and overall survival (OS). We further showed that 4.1B inhibited the proliferation of two GC cell lines, MGC-803 and MKN-45, by impeding the EGFR/MAPK/ERK1/2 and PI3K/AKT pathways. A similar phenotype was also observed in immortalized mouse embryonic fibroblasts (MEF) derived from wild type (WT) and 4.1B knock-out (BKO) mice. Additionally, immunofluorescence (IF) staining and Co-IP showed that protein 4.1B bound to EGFR. Furthermore, the FERM domain of 4.1B interacted with EGFR through the initial 13 amino acids (P13) of the intracellular juxtamembrane (JM) segment of EGFR. The binding of 4.1B to EGFR inhibited dimerization and autophosphorylation of EGFR. Conclusion Our present work revealed that 4.1B plays important regulatory roles in the proliferation of GC cells by binding to EGFR and inhibiting EGFR function through an EGFR/MAPK/ERK1/2 pathway. Our results provide novel insight into the mechanism of the development and progression of GC.


Background
Annually, more than 720,000 people die from gastric cancer (GC) worldwide, making it the second leading cause of cancer-related deaths around the world [1]. Half of all GC cases occur in East Asia (mainly in China), of which a total 677,000 cases occur in developing countries [2]. The incidence of GC is 13.9% in total digestive tract and the trend increases with years [3]. Current therapeutic strategies for GC include mucosectomy by endoscopy, gastrectomy, and/ or chemotherapy. However, the 5-year overall survival is still only 40% [4]. Therefore, more effective treatment options are urgently needed. Understanding the underlying mechanisms that drive GC pathogenesis will facilitate the development of better therapeutic strategies for GC patients.
Erythrocyte Membrane Protein Band 4.1 Like 3 (EPB41L3 or 4.1B) belongs to the protein 4.1 superfamily, of which there are more than 40 members. This superfamily is classified into five subgroups: Protein 4.1 subfamily, ERM proteins, Talin-related molecules, PTPH (protein tyrosine phosphatases) proteins, and NBL4 (novel band 4.1-like 4) proteins based on protein sequence homology [5]. Protein 4.1B together with 4.1R, 4.1G, and 4.1 N belong to the protein 4.1 subfamily and they are encoded by four paralogous genes [6]. The four members of protein 4.1 subfamily contain four highly conserved functional domains: the membrane-binding FERM domain [7][8][9], a FERM-adjacent (FA) regulatory domain [10], a spectrin-actin binding domain (SABD) [11,12], and a C-terminal domain (CTD) unique to the 4.1 proteins [13]. It has been reported that 4.1B is decreased in several cancers, such as renal cell carcinoma [14], ovarian cancer [15], liver cancer [16], and colon cancer [17]. A few studies have addressed the relationship between protein 4.1B and GC [18,19]; however, details of the functional role of 4.1B in GC development and progression have not been unearthed.
Epidermal growth factor receptor (EGFR) belongs to the Erb receptor tyrosine kinase family, of which there are four members: ErbB1 (EGFR), ErbB2 (c-Neu), ErbB3 (HER3), and ErbB4 (HER4) subtypes). EGFR contains 1186 aa and consists of an extracellular ligand-binding domain, a transmembrane domain, and an intracellular domain. Additionally, the intracellular domain is divided into 3 unique segments, namely the juxtamembrane (JM) segment, tyrosine kinase (TK) domain, and a COOH-terminal region [20][21][22]. EGFR plays critical roles in regulating metabolism, growth, and differentiation [23]. Numerous studies have documented EGFR overexpression in tumors, such as squamous carcinoma [24] and some glioblastomas [25]. EGFR has potent mitogenic activity that can either stimulate or inhibit growth of a large variety of normal and malignant cells in vitro.
In this article, we report that 4.1B was downregulated in GC specimens and that 4.1B inhibited GC and MEF cell proliferation through the EGFR/MAPK/ERK1/2 and PI3K/AKT pathways. We further demonstrate that 4.1B bound to EGFR through the interaction of the FERM domain of 4.1B with the P13 of EGFR JM segment. Interestingly, this binding blocked EGFR dimerization and autophosphorylation. Our study provides evidence of the molecular mechanism through which 4.1B inhibits cell proliferation.

Mice
Wildtype C57BL/6 mice were purchased from The Jackson Laboratory. The 4.1B knockout mice have been previously reported [26,27] were kindly provided by Dr. J. Kissil (The-Wistar Institute). These mice were housed in the animal facility of New York Blood Center under specific pathogenfree (SPF) conditions accredited by the American Association for Laboratory Animal Care. Four-week-old athymic BALB/c nude mice used for tumorigenicity assays were purchased from the animal center of Beijing, China. They were housed in the animal room of the fifth affiliated hospital of Zhengzhou University under SPF conditions. All animal experiments were approved by the Institutional Animal Care and Use Committee.

GC cell lines and patient specimens
The GC cell lines MGC-803 and MKN-45 were purchased from Cell Bank of Type Culture Collection of the Chinese Academy of Sciences, Shanghai Institute of Cell Biology. They were cultured in RPMI 1640 medium (HyClone, USA) supplemented with 10% fetal bovine serum (FBS, Invitrogen, USA). All cells were cultured in a humidified incubator at 37°C and 5% CO 2 .
This study included 102 GC patients who underwent gastrectomy from Jan 2011 to Jan 2013. The normal controls were located at least 5 cm away from the tumor. These GC paraffin-embedded tissue sample collections were approved by the Ethics Committee of the fifth affiliated hospital of Zhengzhou University, Henan, China. The 102 patient specimens included 59 males and 43 females. The patient age range was from 25 to 76 years old. Histological grades were classified according to the WHO classification as highly (grade I), moderately (grade II), and poorly differentiated (grade III). The TNM stage was performed according to the AJCC8 classification.

Immortalized MEF cells preparation and culture
Isolation of primary mouse embryo fibroblasts (MEF) from day 13.5 embryos (E13.5) of 4.1B +/+ and 4.1B −/− C57BL/6 mice was performed as previously described [28]. Briefly, the head and internal organs were removed. The remaining embryonic tissues were minced using a pair of scissors and immersed in 0.25% trypsin overnight at 4°C. After 24 h, MEFs were collected after centrifugation at 1500 rpm and maintained in Dulbecco Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS, GIBCO, USA) and 100 μg/ml penicillin/ streptomycin (LEAGENE, China). After two passages, the MEFs were immortalized by retroviral transduction of the SV40 large T antigen. MEFs were cultured in DMEM containing 10% FBS and incubated at 37°C in a humidified environment with 5% CO 2 .

Immunofluorescence
For confocal immunofluorescence microscopy assay, cells were grown on Lab-Tek™ Chambered Coverglass (Thermo Fisher Scientific, USA) pre-coated with 10 μg/ ml fibronectin (Gibco, USA). Cells were fixed with 1% paraformaldehyde (LEAGENE, China) for 15 min and then permeabilized with 0.1% Triton X-100 (Sigma-Aldrich, USA) in 0.25% paraformaldehyde-PBS for 15 min at room temperature, followed by blocking in 10% horse serum (Gibco, USA), 0.1% Triton X-100 in PBS for 30 min to minimize nonspecific antibody binding. Cells were then incubated with primary antibodies at 4°C overnight and then washed with PBS 3 times. This was followed by incubation with the appropriate secondary antibody at room temperature for 30 min. The secondary antibodies were donkey anti-rabbit (Thermo Fisher Scientific, USA) and donkey anti-mouse IgG (Thermo Fisher Scientific, USA) labeled with Alexa Fluor 488 or Alexa Fluor 594. To-pro-3 (Invitrogen, USA) was used to stain the nucleus. Images were collected on a Zeiss LSM510 META confocal microscope (Germany) using × 63 oil-immersion objective. The knockdown efficiencies were detected by Real-time PCR and western blotting after 48 h. The sequences of siRNAs were as follows: human and mouse negative control FAM 5′ -UUCUCCGAACGUGUCACGUTT -3′; human Sp1 5′ -CCAUUAACCUCAGUGCAUUTT -3′; mouse Sp1 5′ -GCGGCAAAGUAUAUGGCAATT-3′.

Western blot analysis
Cells were lysed with RIPA buffer (150 mM NaCl, 25 mM Tris-HCl pH 7.4, 0.1% SDS, 1% Triton X-100, 1% deoxycholate, 2 mM EDTA, Millipore, USA) supplemented with protease inhibitor (Sigma-Aldrich, USA) and phosphatase inhibitor (Roche, Basel, Switzerland) for 30 min on ice. The supernatant was collected by centrifugation with 14,000 rpm and the protein concentration was measured with BCA kit (Thermo Fisher Scientific, USA). Protein samples (50 μg) were separated by 10% SDS-PAGE gels and transferred to nitrocellulose membranes. The membranes were incubated with primary antibodies overnight at 4°C. Membranes were then washed with TBS-T and then incubated with secondary antibodies for 1 h. Signals were detected with ECL kit (Thermo Fisher Scientific, USA) by Chemi Doc™ XRS+ with Image Lab™ Software (Bio-Rad, USA).

Immunohistochemistry
The paraffin-embedded patient specimens were sectioned at 3 μm thickness and mounted on glass slides. After deparaffinization and dehydration with xylene and an alcohol gradient, the slides were washed with PBS. Antigen retrieval was carried out by heating slides for 20 min in citrate buffer (LEAGENE, China). Endogenous peroxidase activity was blocked with 3% hydrogen peroxide and 10% goat serum (Beijing Zhongshanjinqiao Biotechnology Co. LTD, China) for 30 min at room temperature. The slides were incubated with an anti-4.1B primary antibody at 4°C overnight. Biotinylated goat anti-rabbit IgG antibody was then added to the slides for 30 min at 37°C after washing with PBS. Diaminobenzidine (Beijing Zhongshanjinqiao Biotechnology Co. LTD, China) was used to stain the slides for about 1 to 5 min. Slides were then counterstained with hematoxylin (LEAGENE, China) and dehydrated in xylene and mounted with permount.

Tumorigenicity analysis
Five-week-old BALB/C nude mice were injected with 3 × 10 6 of each transfected cell type suspended in 200 μl serum-free RPMI-1640. The cells were inoculated into the subaxillary region for the tumorigenic assay. Tumors were palpable after 1 week and monitored every 3 days. The tumor size was measured with a Vernier caliper and tumor volume was calculated as follows: volume = 1/ 2 × (long axis) × (short axis) 2 . Experimental mice were euthanized after 2 weeks. The resected tumors were fixed with 10% formalin, embedded in paraffin, and sectioned (3 μm). Sections were stained with hematoxylin and eosin (H&E) (LEAGENE, China) and Ki-67 (LEA-GENE, China) for light microscopy examination.

Co-immunoprecipitation
MEFs were lysed with ice-cold lysis buffer (50 mM HEPES, pH 8.3, 420 mM KCl, 0.1% NP-40, 1 mM EDTA) supplemented with a proteinase inhibitor cocktail (Roche, Basel, Switzerland) for 30 min on ice. Supernatants were collected after centrifugation at 15,000 g at 4°C for 10 min and protein concentration was determined by the Bradford method using BSA as a standard (Bio-Rad, USA). Protein extracts (500 μg per sample) were incubated with either 5 μg anti-4.1B-HP or anti-EGFR antibody or preimmune IgG in 500 μl of Co-IP buffer (Active motif, CA) at 4°C overnight with rotation. The immunoprecipitates were isolated using Protein-G beads (Millipore, USA) and separated by 10% SDS-PAGE and then transferred to a nitrocellulose membrane. The membrane was probed with antibodies against EGFR or 4.1B-HP.

Pull-down assay
GST-tagged recombinant different fragment cytoplasmic domain of EGFR and various functional domains of 4.1B were coupled to glutathione-Sepharose-4B beads at room temperature for 30 min. Immobilized streptavidin beads were mixed with biotinylated 13 EGFR peptide prior to incubation with GST-tagged 4.1B domains. Beads were pelleted and washed. His-tagged 4.1B with different GSTtagged cytoplasmic domain of EGFR and GST-tagged 4.1B domains with biotinylated 13 EGFR peptide were added to the coupled beads in a final volume of 100 μl. The final concentration of the coupled protein was 2 μM. The mixture was incubated for 1 h at room temperature, pelleted, washed and eluted with 10% SDS. The pellet was analyzed by SDS-PAGE. The binding of His-tagged 4.1B domains with EGFR intracellular fragments was detected by western blot using anti-His antibody. The binding of GSTtagged 4.1B domains with 13 EGFR peptide was detected by western blot using anti-GST antibody. GST was used as negative control in all experiments.

Statistics
All statistical analyses were performed by using SPSS 20.0 software. Difference between 2 groups was determined by Student t test orχ 2 test. DFS and OS were analyzed by Kaplan-Meier analysis with the log-rank test applied for comparison. And the survival data were examined by univariate and multivariate COX proportional hazards model. Variables with a value of p < 0.05 in univariate were checked in multivariate analysis by COX regression analyses. And two-sided p < 0.05 was considered statistical significance.

Expression of 4.1B in GC patients and GC cell lines
To explore the relationship between 4.1B and GC, we analyzed 4.1B expression in 102 gastric adenocarcinoma patient samples by immunochemistry. The results revealed that 4.1B was significantly different between tumor tissues and their matched adjacent non-tumor tissues ( Fig. 1a; Table 1; χ 2 = 32.23; p < 0.001). There was strong expression of 4.1B in the cell membrane and cytoplasm of the majority of adjacent non-tumor tissues (91.17%) (Fig. 1a). In contrast, tumor tissues showed little to no expression of 4.1B (Fig. 1a). There was a negative correlation between 4.1B expression level with tumor size, pathologic differentiation, lymph node metastasis and TNM stage ( Table 2). There was no significant association between 4.1B expression and patients age, gender, Helicobacter pylori infection, tumor location, WHO classification, vascular invasion, depth of invasion and neural invasion ( Table 2).
To further explore the correlation of 4.1B and prognosis, we analyzed the median survival about the disease-free survival (DFS) and overall survival (OS) by Kaplan-Meier analyses and log-rank test. Seven specimens were excluded because of accidental death or lost to follow-up. The DFS results showed the 5-year median survival of 4.1B positive patients was 22 months, while the 4.1B negative specimens was 15 months. The 5-year median survival about OS is 51 months in 4.1B positive patients, while the 4.1B negative specimens was 33 months. This result showed that 4.1B positive GC patients had better survival time than negative ones ( Fig. 1b and c; p < 0.001).
To further evaluate whether 4.1B is an independent factor for GC patient prognosis, we performed the univariate and multivariate COX regression analyses for DFS and OS. The results showed the 4.1B expression, tumor size, lymph metastasis (N0 vs. N1 + N2 + N3) and TNM analyses (I + II vs. III) were the prognostic factor.  Table 3).
We also investigated 4.1B protein expression in two GC cell lines with starkly different rates of proliferation ( Fig. 2A). There was a clear detection of a prominent protein band at 130 kDa in the MKN45 cell line. However, no such band was detected in the MGC-803 cell line ( Fig. 2A). The mRNA level of 4.1B was also consistent with the protein result by real-time PCR (data not shown). These results suggest that 4.1B was inversely associated with the proliferation of GC cell lines.

4.1B suppresses GC cell proliferation in vitro
To further explore the effect of 4.1B in GC cell lines, we transiently transfected MGC-803 cells with pEGFP-4.1B to exogenously increase 4.1B expression. Conversely, 4.1B double nickase plasmid was transfected transiently to silence the expression of endogenous 4.1B. Real-time PCR and western blot analysis confirmed successful overexpression at the mRNA and protein level and knockout at protein level ( Fig. 2B-a and b; C-a). Figure 2B-d showed that 4.1B overexpression suppressed proliferation of MGC803 in comparison to pEGFP-C3 control at different time points (p < 0.001). Conversely, proliferation was promoted in 4.1B-silenced MKN45 cells (Fig. 2C-b; p < 0.001).
Collectively, these results demonstrated that 4.1B can suppress GC cell proliferation. These results suggest that 4.1B can act as a tumor suppressor gene in GC cells.  (Fig. 3c and g; p < 0.001and p < 0.05). The mitotic ability, as shown by Ki-67 staining, was suppressed in 4.1B-sufficient cells (Fig. 3d, h).

4.1B suppresses GC cell proliferation by affecting the EGFR/MAPK/ERK1/2 and PI3K/AKT pathways
Previous studies have focused on the relationship between 4.1B and cell differentiation [17], adhesion [30], motility [31], and apoptosis [32]. There were also some studies on 4.1B and cell proliferation, but these studies were limited to a characterization of cell cycle proteins [33]. We wanted to further explore the mechanisms by which 4.1B affects cell proliferation.
Cell proliferation is commonly regulated by the EGFR/ MAPK and PI3K/AKT pathways. Therefore, we examined changes in these two key signaling pathways by western blot. Figure 4 showed that 4.1B deficiency resulted in markedly increased levels of ERK1/2 and AKT phosphorylation. Total ERK1/2 and AKT protein levels remained unchanged. 4.1B did not affect p38 and JNK phosphorylation or their corresponding total protein levels. Interestingly, we observed that 4.1B loss resulted in an increase in the protein level of EGFR (Fig. 4).

Deficiency of 4.1B leads to hyperproliferation of MEF cells due to increased EGFR expression
We then examined the proliferation of immortalized MEFs derived from wild type and 4.1B knockout mice. Figure 5A shows increased proliferation of 4.1B knock-out MEF cells. The increased proliferations were accompanied by the increased phosphorylation of ERK1/2, AKT and EGFR (Fig. 5B). Interestingly, while there were no changes in protein levels of ERK1/2 and AKT, total EGFR protein level was also upregulated in immortalized 4.1B KO MEFs (Fig. 5B). These findings suggest that 4.1B may affect cell proliferation by regulating EGFR. The finding that EGFR    Figure 5D shows negative correlation between 4.1B expression and EGFR mRNA levels in various cell types, demonstrating that deficiency of 4.1B leads to increased EGFR transcription.
To further define mechanisms for the increased mRNA level of EGFR, we examined the expression of Sp1, the transcription factor for EGFR. The data in Fig.   5E shown that 4.1B deficiency led to increased expression of Sp1. Interestingly, knockdown of Sp1 in 4.1B-deficient cells led to decreased EGFR expression at mRNA and protein level (Fig. 5F).
Given the consistency in the data between MEFs and GC cell lines, we used MEFs as the experimental model in subsequent assays.

Association of 4.1B with EGFR in MEF cells
We next examined the association of 4.1B with EGFR in MEF cells. Figure 6a shows that while GFP was diffusely  Fig. 6b shows that endogenous EGFR was pulled down by an anti-4.1B HP antibody and that endogenous 4.1B was pulled down by an anti-EGFR antibody. These findings suggest that 4.1B binds to EGFR.

Discussion
The results of our study have highlighted a novel function for 4.1B as a tumor suppressor, which inhibits proliferation of gastric cancer cells. Additionally, we demonstrate the details of the sites involved in interaction between 4.1B and EGFR. This study is the first to document the mechanism by which 4.1B inhibits tumor cell proliferation by binding to the JM segment of EGFR. Previous studies showed that different cancers express little to no 4.1B [14][15][16][17] due to promoter hypermethylation [14,19,[34][35][36]. Only a few studies have assessed 4.1B in GC [18,19]. Our study examined the correlation of 4.1B with GC clinical parameters. The IHC results showed that 4.1B was mainly expressed in the cell plasma membrane and was downregulated in GC adenocarcinoma. Our clinical data also demonstrated that 4.1B can affect tumor size, pathologic differentiation, vascular invasion, and TNM stage. The expression of 4.1B is also associated with patient overall survival (OS). These results suggest that 4.1B may serve as a tumor suppressor in gastric cancer.
Several studies have alluded to the molecular mechanisms underlying the oncogenic effect of 4.1B deficiency. Sakurai-Yageta et al. reported that 4.1B regulated CADM1 to affect epithelial cell adhesion. CADM1 associates with 4.1B/DAL-1 through the 4.1B binding motif, while loss of the CADM1-4.1B/DAL-1 complex may affect cancer cell adhesion and enhance cancer cell invasion and/or metastasis [30]. Other studies have shown that 4.1B/DAL-1 can anchor F-actin to the cell membrane, thereby inhibiting cell motility by supporting orderly arrangement of actin stress fibers. Loss of the 4.1B/DAL-1 complex may promote F-actin reorganization and enhance cell motility, which in turn, may increase tumor migration [15]. Other studies have also reported 4.1B regulation of cell mobility through the activity of adhesion molecules [15,30,37,38].
In this study, we focused on the functional role of 4.1B in gastric cancer cell proliferation. Although studies have examined the relationship of 4.1B and GC [18,19], there have been no reports of the functional effect of 4.1B on GC cell proliferation. We investigated the relationship between Src, Rac1, MLK3, and JNK, resulting in reduced expression cyclin A and decreased retinoblastoma (Rb) protein hyperphosphorylation [33,39,40].
We found that 4.1B blocked GC cell line proliferation through the proto-oncogenic EGFR/MAPK/ERK1/2 and Fig. 7 4.1B FERM domain binds to EGFR JM segments. a Sketch map of intracellular EGFR design information. GST-tagged three fragments of intracellular EGFR: EGFR fragment with (amino acids fragment 644-955 (EGFR1)) and without (amino acids fragment 956-1186 (EGFR2)) JM fragments and JM fragment deletion the first thirteen amino acids (amino acids fragment 657-955 (EGFR1△aa13)). The black arrow shows EGFR fragment (amino acids fragment 644-955 (EGFR1)) with two or three amino acids mutated randomly: aa13-RRRHIVRKRTLRR, EGFR P13(RRR-AAA)-AAAHIVRKRTLRR, EGFR P13(RKR-AAA)-RRRHIVAAATLRR, EGFR P13(RR-AA)-RRRHIVRRRTLAA. AAA means mutated region. b 4.1B binds to EGFR fragments with the first thirteen amino acids of JM fragment by pull-down assay. c Sketch map of domains of 4.1B and the FERM domain of 4.1B binds to EGFR by pull-down assay. d Conclusions of 4.1B FERM domain interacts with EGFR fragment with the first thirteen key amino acids of JM fragment PI3K/AKT signaling pathways. Of note, we observed that perturbing 4.1B expression in GC cell lines resulted in a change in the levels of total EGFR. Similar effects on proliferation and EGFR levels were observed in immortalized 4.1B deficiency MEFs.
We used an EGFR inhibitor to address our hypothesis that 4.1B can inhibit EGFR-mediated cell proliferation in immortalized 4.1B deficiency MEFs. Our results also showed that 4.1B can suppress EGFR mRNA levels.
Activation of EGFR requires more than just ligand engagement to the extracellular EGFR segment. EGFR dimerization also requires coupling of the transmembrane domain and JM segment [41]. The initial 13 amino acids (P13) within the intracellular JM region (R645-R657) are required for proper dimerization and autophosphorylation of EGFR [42]. 4.1B is localized to the plasma membrane and is distributed along cell-cell junctions [26], as well as cell-basement membrane contacts [33]. Therefore, we hypothesized that 4.1B could bind to the P13 region of the EGFR JM segment to block EGFR activation. Our IF staining and co-IP assay demonstrated that 4.1B can indeed, directly bind to EGFR. Our pull-down assay clearly showed 4.1B interaction with the P13 region of the EGFR JM fragment. We further demonstrated that the FERM domain of 4.1B can bind to the P13 region of the EGFR JM segment.
Our schematic diagram (Fig. 8) explains clearly the mechanism of 4.1B blocking cells proliferation by inhibiting the EGFR synthesis. The combination of protein 4.1B with P13 of EGFR JM segments blocks the binding of EGFR monomers intracellular JM segments. This blocking hinders EGFR monomers dimerization and autophosphorylation, and the conformational coupling is stopped. Thus, EGFR activation and autophosphorylation is suppressed in 4.1B-sufficient cancer cells. This can explain why the phosphorylated EGFR was down-regulated in our transfected GC cells and MEF cells with protein 4.1B existence. The data showed that phosphorylated ERK1/2 was down-regulated by inhibition of EGFR/Ras/Raf/ERK1/2 pathway.

Conclusion
Our findings are significant and novel. First, our results clearly showed that 4.1B functions as a tumor suppressor in GC. Our study is also the first report of 4.1B inhibition of GC cell proliferation by binding to EGFR monomers. This is also the first demonstration of the precise domains of 4.1B and EGFR that facilitate their interaction. Given the importance of EGFR signaling in cancer cell growth and tumor progression, our study highlights a potential new therapeutic target for gastric cancer. Our data suggest that 4.1B-targeting may be a viable strategy to combat tumor progression in gastric cancer patients.